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Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation.

Watson RT, Shigematsu S, Chiang SH, Mora S, Kanzaki M, Macara IG, Saltiel AR, Pessin JE - J. Cell Biol. (2001)

Bottom Line: Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10.We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains.These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft-localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

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Endogenous TC10 colocalizes with caveolin-positive structures in the adipocyte plasma membrane. Differentiated 3T3L1 adipocytes were left untreated and either the intact cells were directly fixed (a–c), or used to isolated plasma membrane sheets (d–f) as described in Materials and methods. The samples were then colabeled with a polyclonal TC10 antibody (a and b) and a monoclonal caveolin 1 antibody (b and e) and subjected confocal fluorescent microscopy. The merged images are presented in panels c and f. These are representative fields from three independent determinations. Bar, 10 μM.
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fig4: Endogenous TC10 colocalizes with caveolin-positive structures in the adipocyte plasma membrane. Differentiated 3T3L1 adipocytes were left untreated and either the intact cells were directly fixed (a–c), or used to isolated plasma membrane sheets (d–f) as described in Materials and methods. The samples were then colabeled with a polyclonal TC10 antibody (a and b) and a monoclonal caveolin 1 antibody (b and e) and subjected confocal fluorescent microscopy. The merged images are presented in panels c and f. These are representative fields from three independent determinations. Bar, 10 μM.

Mentions: To determine whether the endogenous TC10 protein was localized to plasma membrane lipid raft microdomains, adipocytes were first colabeled with monoclonal caveolin and polyclonal TC10 antibodies. In intact cells, the endogenous TC10 protein was predominantly confined to the plasma membrane with very little detectable expression on intracellular membranes (Fig. 4 , a and c). Similarly, caveolin displayed strong plasma membrane localization with lower, but still detectable, levels in the perinuclear region (Fig. 4, b and c). We also prepared plasma membrane sheets and examined the localization patterns of TC10 and caveolin. Although individual caveolae (50–80 nm) are too small to be detected by laser confocal microscopy, caveolae can form large clusters or torus-shaped structures in adipocytes (Gustavsson et al., 1999). Endogenous TC10 was found to colocalize exceptionally well with caveolin in these distinct ring-like domains at the plasma membrane (Fig. 4, d–f).


Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation.

Watson RT, Shigematsu S, Chiang SH, Mora S, Kanzaki M, Macara IG, Saltiel AR, Pessin JE - J. Cell Biol. (2001)

Endogenous TC10 colocalizes with caveolin-positive structures in the adipocyte plasma membrane. Differentiated 3T3L1 adipocytes were left untreated and either the intact cells were directly fixed (a–c), or used to isolated plasma membrane sheets (d–f) as described in Materials and methods. The samples were then colabeled with a polyclonal TC10 antibody (a and b) and a monoclonal caveolin 1 antibody (b and e) and subjected confocal fluorescent microscopy. The merged images are presented in panels c and f. These are representative fields from three independent determinations. Bar, 10 μM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196453&req=5

fig4: Endogenous TC10 colocalizes with caveolin-positive structures in the adipocyte plasma membrane. Differentiated 3T3L1 adipocytes were left untreated and either the intact cells were directly fixed (a–c), or used to isolated plasma membrane sheets (d–f) as described in Materials and methods. The samples were then colabeled with a polyclonal TC10 antibody (a and b) and a monoclonal caveolin 1 antibody (b and e) and subjected confocal fluorescent microscopy. The merged images are presented in panels c and f. These are representative fields from three independent determinations. Bar, 10 μM.
Mentions: To determine whether the endogenous TC10 protein was localized to plasma membrane lipid raft microdomains, adipocytes were first colabeled with monoclonal caveolin and polyclonal TC10 antibodies. In intact cells, the endogenous TC10 protein was predominantly confined to the plasma membrane with very little detectable expression on intracellular membranes (Fig. 4 , a and c). Similarly, caveolin displayed strong plasma membrane localization with lower, but still detectable, levels in the perinuclear region (Fig. 4, b and c). We also prepared plasma membrane sheets and examined the localization patterns of TC10 and caveolin. Although individual caveolae (50–80 nm) are too small to be detected by laser confocal microscopy, caveolae can form large clusters or torus-shaped structures in adipocytes (Gustavsson et al., 1999). Endogenous TC10 was found to colocalize exceptionally well with caveolin in these distinct ring-like domains at the plasma membrane (Fig. 4, d–f).

Bottom Line: Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10.We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains.These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft-localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

Show MeSH
Related in: MedlinePlus